In plants, two classes of genes encode starch branching enzymes, known respectively as BEI, and BEII. In the monocotyledonous cereals, there is strong evidence demonstrating that the BEII class contains two independent types of genes, known in maize as BEIIa and BEIIb (Gao et al., 1996; Fisher et al., 1996). In barley, two types of genes have been reported, and shown to be differentially expressed (Sun et al., 1998), An additional class of branching enzyme (50/51 kD) from barley has also been described (Sun et al., 1996).
In dicotyledonous plants, loss of BEII activity through either mutation (Bhattacharyya et al., 1990) or gene suppression technologies gives rise to starches containing high amylose levels (Safford, 1998, Jobling 1999).
In monocotyledonous plants, mutations giving rise to high amylose contents are known in maize, rice and barley. In neither rice (Mizuno et al., 1993) nor barley (Schondelmaier et al., 1992) have the known high amylose phenotypes been associated with the BEIIa or BEIIb mutations respectively. However, in maize it is firmly established that the high amylose phenotype is associated with down regulation of the BEIIb gene (Boyer et al., 1980; Boyer and Preiss, 1981, Fisher et al, 1996).
The impact of down-regulation of BEI has been investigated through antisense inhibition in potato tuber; the down-regulation has been found to alter the properties of the starch, but not its gross structural features, such as the amylose content (Filpse et al., 1996). In wheat, antisense down-regulation of BEI activity has small but significant effects on starch structure (Baga et al, 1999). The branching enzyme I gene from maize has been cloned (Kim et al., 1998), but mutants affecting branching enzyme I activity in maize are not known.
No mutations specifically reducing BEIIa activity have been reported, and no gene suppression experiments in plants have succeeded in reducing BEIIa activity, although the du1 mutation in maize is known to reduce the expression of both BEIIa and starch synthase III. However, the du1 mutation is now known to be due to mutation of the structural gene for starch synthase III (Gao 1998, Cao 1999).
In our previous patent application No. PCT/AU98/00743 (WO99/14314), we have described the structure of a BEII gene from wheat, which we have subsequently designated the BEIIa gene.
In the present application we describe the isolation of a second BEII gene from wheat, which we have designated the BEIIb gene, and discuss the uses to which this gene sequence can be applied. We have surprisingly found that in wheat the expression level of the various branching enzymes is very different to that in maize and barley. In this specification we show that BEIIb in wheat is expressed at low levels in the soluble fraction of the wheat endosperm, and is predominantly found within the starch granule. This indicates that there are important differences in the regulation of gene expression in wheat compared to other cereals, suggesting that the manipulation of the amylose to amylopectin ratio in wheat will involve the manipulation of more than just the BEIIb gene.
We have also surprisingly found that the BEIIa and BEIIb gene structures are highly conserved with respect to exon size and position, allowing us to prepare DNA-based diagnostics which they can distinguish not only the BEIIa and BEIIb classes of genes, but also the forms of these genes encoded on the A, B and D genomes of wheat, and to identify the BEIIb proteins expressed by the wheat A, B and D genomes, providing an essential tool for the screening of wheat germplasm for null or altered alleles of wheat branching enzyme IIa.